Recurrent pregnancy losses are a hallmark of the antiphospholipid syndrome (APS), a complicated major hypercoagulable disorder without an established mechanism that is currently diagnosed with phenomenologic surrogate tests. APS is associated with a host of thrombotic complications including recurrent pregnancy losses, stroke, deep vein thrombosis and pulmonary embolism. The placental anticoagulant protein, annexin A5 (AnxAS) is a potent phospholipid (PL)-binding protein that forms 2-dimensional anticoagulant crystal shields over membranes containing anionic PLs. AnxAS is highly expressed on the apical membranes of placental syncytiotrophoblasts where it is in an anatomic position to promote blood fluidity in the intervillous space. The core hypothesis of this application is that pathogenic antiphospholipid (aPL) antibodies bind to specific domains on (32-glycoprotein I, the major cofactor for aPL antibodies. These results in formation of macromolecular antibody-antigen complexes on the apical surfaces of syncytiotrophoblasts that create defects in the AnxAS anticoagulant shield and thereby promote coagulation reactions and reduce blood fluidity in the placental circulation. We have provided strong evidence for this hypothesis and have begun to develop innovative treatments that target this disease mechanism and mechanistic tests that identify resistance to AnxAS anticoagulant activity. This grant will extend the above findings with the following specific aims: 1) To investigate the effects of aPL antibodies on the AnxAS anticoagulant shield that is present on the apical membranes of placental trophoblasts. 2) To translate the findings of the first specific aim into innovative treatments targeting this disease mechanism and into mechanistic assays to diagnose the disease. These aims will be accomplished with atomic force microscopy, ellipsometry, binding studies, and coagulation enzyme studies using PL bilayers, cultured placental cells and placental villi and translational tests for AnxAS resistance. This project will be a major contribution toward elucidating a novel mechanism for pregnancy losses in a significant disease which is lacking an established pathophysiology and will open new approaches toward mechanistically-based diagnosis and treatment.